A new gene inventory of the ubiquitin and ubiquitin-like conjugation pathways in Giardia intestinalis

Abstract

BACKGROUND Ubiquitin (Ub) and Ub-like proteins (Ub-L) are critical regulators of complex cellular processes such as the cell cycle, DNA repair, transcription, chromatin remodeling, signal translation, and protein degradation. Giardia intestinalis possesses an experimentally proven Ub-conjugation system; however, a limited number of enzymes involved in this process were identified using basic local alignment search tool (BLAST). This is due to the limitations of BLAST's ability to identify homologous functional regions when similarity between the sequences dips to < 30%. In addition Ub-Ls and their conjugating enzymes have not been fully elucidated in Giardia. OBJETIVE To identify the enzymes involved in the Ub and Ub-Ls conjugation processes using intelligent systems based on the hidden Markov models (HMMs). METHODS We performed an HMM search of functional Pfam domains found in the key enzymes of these pathways in Giardia's proteome. Each open reading frame identified was analysed by sequence homology, domain architecture, and transcription levels. FINDINGS We identified 118 genes, 106 of which corresponded to the ubiquitination process (Ub, E1, E2, E3, and DUB enzymes). The E3 ligase group was the largest group with 82 members; 71 of which harbored a characteristic RING domain. Four Ub-Ls were identified and the conjugation enzymes for NEDD8 and URM1 were described for first time. The 3D model for Ub-Ls displayed the β-grasp fold typical. Furthermore, our sequence analysis for the corresponding activating enzymes detected the essential motifs required for conjugation. MAIN CONCLUSIONS Our findings highlight the complexity of Giardia's Ub-conjugation system, which is drastically different from that previously reported, and provides evidence for the presence of NEDDylation and URMylation enzymes in the genome and transcriptome of G. intestinalis. Key words: ubiquitin-ubiquitin-like protein-ubiquitin ligase-deubiquitinating enzymes-Giardia-hidden Markov models Ubiquitin (Ub) and ubiquitin-like modifiers (Ub-Ls) are small proteins that covalently attach to protein substrates and regulate various cellular processes such as the cell cycle, endocytosis, signaling pathways, intracellular trafficking, DNA repair and transcription, among others. (1) Ub and Ub-Ls share two common features: a β-grasp fold composed of a five-stranded β-sheet and a C-terminal diglycine motif (GG) used for conjugation to target proteins. Currently, the Ub-L family includes 10 members: small ubiquitin modifier (SUMO), neural precursor cell expressed developmentally downregulated 8 (NEDD8) or Related to Ubiquitin 1 (RUB 1) in yeast, Ubiquitin-Related Modifier-1 (URM1), Ubiquitinfold Modifier 1 (UFM1), autophagy-related proteins 8 and 12 (ATG8 and ATG12), interferon-stimulated gene 15 (ISG15), human leukocyte antigen (HLA)-F adjacent transcript 10 (FAT10), fan ubiquitin-like protein 1 (FUB1), and histone mono-ubiquitination 1 (HUB1). (2) The first step in the Ub-conjugation cascade is activation, which is mediated by the E1 protein (UBA1 in the b...

Functional Differentiation of Cyclins and Cyclin-Dependent Kinases in Giardia lamblia

“…Recently, protein small ubiquitin-like modifier (SUMO)ylation was reported to be involved in diverse cell cycle-related processes in Giardia ( 40 ). Genes encoding this pathway are conserved in Giardia ( 41 , 42 ), and SUMOylation of arginine deiminase has been found to be involved in the encystation of Giardia ( 43 , 44 ). Use of the GPS-SUMO 1.0 tool predicted potential sites of SUMOylation in Glcyclins 3977, 14488, 17505, and 22394 (data not shown).…”

Ubiquitin-Like Modifiers: Emerging Regulators of Protozoan Parasites

“…The presence of genes encoding SUMO and SUMO-specific enzymatic machinery [ 21 , 92 ], as well as cytoplasmic and nuclear localisation of this Ubl, have been reported for another flagellate protozoan, G. lamblia , in the motile trophozoite form [ 92 ]. Knockdown of Gl SUMO led to alteration of trophozoite cell morphology and distortion of the ventral adhesive disk, a unique and essential structure that enables attachment of the parasite to the intestinal mucosa of the host.…”

“…The Ubl family includes neural precursor cell-expressed developmentally downregulated 8 (NEDD8), small ubiquitin-related modifier (SUMO), ubiquitin-fold modifier 1 (UFM1), ubiquitin-related modifier 1 (URM1), autophagy-related proteins 8 and 12 (ATG8 and ATG12), interferon-stimulated gene 15 (ISG15), and human leukocyte antigen-F adjacent transcript 10 (FAT10) [ 18 ]. A rich variety of Ubls have been found in parasitic protozoa [ 19 , 20 , 21 , 22 ], with the exception of ISG15 and FAT10 ( Table 1 ). These two modifiers have been implicated in immune and stress responses in the context of multicellular organisms [ 23 , 24 ], which may explain their absence in unicellular parasitic protozoans.…”

“…In particular, algorithms based on amino acid sequence motifs [ 36 , 37 ], protein secondary structure [ 38 , 39 ], or a combination of both [ 40 ], have been devised to determine SUMOylation sites. Enabled by the release of protozoan proteomic and genomic datasets over the last two decades [ 41 , 42 , 43 ], computational approaches have been instrumental in identifying putative Ubls and enzymes mediating Ubl cascades in these organisms based on sequence and structural similarity to relevant conserved domains [ 19 , 20 , 21 , 22 ]. However, due to significant divergence from model organisms and a high proportion of proteins without any known homology, functional studies are critical to elucidating these pathways in parasitic protozoa.…”